面對大量廢棄蛋白副產物的時代，及大量氮肥料施用於土壤中，許多優養化河川溪流相關問題隨之而出，目的為提高轉化環境中含氮廢棄物，我們在此提供一株具有潛力於極度鹼性環境中分解蛋白質之菌株，進而轉化為有用之色胺酸，此胺基酸常是神經作用相關藥物之前驅物。
在本論文研究方向為利用廢棄大腸桿菌菌體做為蛋白營養液來源，嗜鹼性Bacillus marmarensis是一隻還未完善開發之菌株，先以木糖及葡萄糖測試其偏好氧含量環境，分析其基本生長狀況下之有機酸產物。嘗試各種前處理方法來取得菌體蛋白養分，探討芽孢桿菌蛋白酶分解能力、蛋白營養程度和其他變因之關係。測試對於生產菌株之各種色胺酸類似物毒性，選擇有效之類似物符合後續之要求，利用NTG藥物突變生產菌株及天然競爭篩選方式選出目標菌株，再經由調整緩衝液之pH值來克服實驗障礙，藉而篩選出有色胺酸氨基酸生產能力之菌株，最高色胺酸產量為75.2mg/L，最後嘗試將其進行轉殖表現異源基因corynebacterium glutamicum之基因cFMO。

Faced with the era of a large number of waste protein by-products, and the application of a large amount of nitrogen fertilizer to the soil, many problems related to the eutrophication of rivers and streams have followed. With the aim of increasing the value of the nitrogenous waste in the environment, we provide a strain that has the potential to degrade proteins into protein skeleton in an extremely alkaline environment and then converted into valuable biochemical such as tryptophan which is often a precursor to neurologically-related drugs without the need for sterility.
In this paper, the research direction is to use the waste E. coli bacteria as the source of protein nutrient solution. The alkaliphile Bacillus marmarensis is a strain that has not been fully developed. We test its preferred oxygen content environment based on xylose and glucose. The organic acid product under growth conditions. we performed a variety of pre-treatment methods to obtain cell lysates of the model microorganism E. coli.We explored the effect of protein source, oxygen level, and pH on the hydrolytic ability of the B. marmarensis protease. In order to improve L-tryptophan production titers, we will expose B. marmarensis to various amino acid analogs and perform a selection process coupled to NTG random mutagenesis and natural passing with increasing tryptophan analog concentration. Then, by adjusting the pH value of the buffer to overcome the experimental obstacles, the tryptophan amino acid production ability of the strain was screened, and the highest tryptophan yield was 75.2 mg/L. Finally ,we attempts to transform it to express the gene cFMO of the heterologous gene corynebacterium glutamicum.

目錄
摘要 I
Abstract II
謝誌 III
目錄 IV
圖表 VI
1 文獻回顧 1
1-1研究背景及目的 1
1-2 前處理簡介 5
1-3 嗜鹼性Bacillus marmarensis 6
1-4 色胺酸和其類似物 11
1-5 N- 甲基 -N'- 硝基 -N- 亞硝基胍介紹 14
2 實驗材料及方法 16
2-1 化學藥品及試劑 16
2-2 菌株介紹 16
2-3 實驗之培養基 16
2-3-1 琥珀酸與乳酸生產試驗 16
2-3-2 生產色胺酸的前處理蛋白培養液 16
2-4 琥珀酸與乳酸之生產條件 17
2-5 不同pH、濃縮之蛋白汁液營養程度探討 17
2-6 色胺酸類似物毒性之生產條件 18
2-7 NTG毒性及配合色胺酸類似物篩選之過程條件 18
2-8 酵素分析活性測試 18
2-9 轉殖質體於Bacillus marmarensis 19
2-10 代謝產物分析方法 19
2-11 本論文使用菌株之質體及引子 20
3 實驗結果 22
3-1 B. marmarensis基本生長狀況概況探討 22
3-1-1 B. marmarensis不同碳源(葡萄糖/木醣)生長之產物探討 22
3-1-2 不同氧氣濃度培養條件的探討 23
3-2 B. marmarensis用前處理大腸桿菌廢棄菌體作為營養源 25
3-2-1 大腸桿菌之不同前處理方式探討 25
3-3 以質體pNW33N成功地轉殖B. marmarensis 27
3-3-1 以質體pCC06/pCC07嘗試轉殖B. marmarensis 28
3-3-2 質體 pCC07t(額外含tnaA基因)轉殖B. marmarensis 30
3-3-3 帶有質體 pCC07之 B.marmarensis進行酵素分析 31
3-3-4 以GFP螢光基因蛋白檢測 B. marmarensis之啟動子 32
3-4以NTG藥物/天然馴化繼代搭配色胺酸類似物篩選突變B. marmarensis菌株 34
3-4-1 不同色胺酸類似物濃度之毒性測試 34
3-4-2 以藥物NTG突變B. marmarensis菌株之探討 36
3-4-3 以蛋白質前處理液搭配7-Azatryptophan類似物天然馴化繼代菌株B. marmarensis 39
4 結論 40
5 參考文獻 41

圖表
圖1-1-1實驗流程圖 1
圖1-1-2氮循環示意圖 2
圖1-1-3廢棄物回收價格以及處理後價格 4
圖1-1-4廢棄物處理狀況 4
圖1-3-1 B. marmarensis在鹼性環境下之狀況 8
圖1-3-2 B. marmarensis在蓄意污染之培養物16s rDNA菌種證明其優勢性 9
圖1-3-3基於16S rRNA之基因序列，近鄰結合法(研究DNA而建立親緣關係)顯示菌株GMBE 72T之位置 10
圖1-4-1蛋白質合成的準確度；aminoacyl-tRNA合成酶校正機制 11
圖1-4-2 tyrosyl-tRNA合成酶結合和激活酪氨酸或酪氨酸類似物 12
圖1-4-3 Bacillus subtilis中之芳香族氨基酸回饋抑制示意圖 13
圖1-4-4 Bacilus subtilis在不同色氨酸類似物之葡萄糖營養液情形 14
圖1-5-1 NTG隨不同pH及作用時間分解之狀況 15
圖3-1-1 B. marmarensis生產菌株於添加不同碳源之生長曲線圖 22
圖3-1-2生產菌株於添加不同碳源生產有機酸之分析結果 23
圖3-1-3生產菌株於不同含氧比生產有機酸之分析結果 24
圖3-2-1 B. marmarensis及大腸桿菌在不同pH值前處理下之差異性 25
圖3-2-2 B. marmarensis生長變因探討 26
圖3-3-1 作為放大質體之宿主e. coli XL1 Blue順利表現出深藍色 28
圖3-3-2 質體pCC07轉殖進入B. marmarensis之塗盤狀況 28
圖3-3-3 質體pCC06/07搭配啟動子1361/3358之設計圖 29
圖3-3-4 放大質體之宿主e. coli XL1順利表現呈深藍色(左)而B. marmarensis卻 沒有顏色變化(右) 30
圖3-3-5 質體pCC07t轉殖完成之B. marmarensis基因分兩段A及B 30
圖3-3-6 質體pCC07t搭配啟動子3358及tnaA/cFMO基因之設計圖 31
圖3-3-7 10ul /20ul crude extract於5mM indole環境消耗cofactor之變化量 32
圖3-3-8 作為放大質體之宿主大腸桿菌順利表現螢光(左)而B. marmarensis卻沒有任何變化(右) 32
圖3-3-9 質體pCCAgfp轉殖完成之B. marmarensis及作為放大質體之宿主大腸桿菌基因分段A及B段 33
圖3-3-10 質體pCCAgfp搭配啟動子1361之設計圖 33
圖3-4-1 B. marmarensis於不同色胺酸類似物之毒性測試 34
圖3-4-2 B. marmarensis於不同色胺酸類似物之毒性測試 35
圖3-4-3 B. marmarensis於不同色胺酸類似物之相對生長比 35
圖3-4-4 B. marmarensis於不同pH緩衝液下洗滌後NTG 0/30min及有無在冰上操作之塗盤測試 36
圖3-4-5 B. marmarensis於不同pH緩衝液下洗滌後NTG 0/30min及有無在冰上操作之塗盤測試 36
圖3-4-6 B. marmarensis於pH9緩衝液下洗滌後NTG 0/30min及有無在冰上操作之塗盤測試 37
圖3-4-7 B. marmarensis於pH8.5/10緩衝液下稀釋塗盤狀況 38
圖3-4-8 B. marmarensis於pH10檸檬酸鹽緩衝液下洗滌後NTG 60min在冰上操作之蛋白前處理液繼代多次實驗(7-Azatryptophan) 38
圖3-4-9 B. marmarensis於200/ 500/ 800/ 1000 mg/L 7AzaTrp類似物之蛋白前處理液繼代多次實驗(7-Azatryptophan) 39
圖3-4-10 B. marmarensis以蛋白質前處理液天然馴化及NTG突變後隨機挑選之單一菌落於16小時色胺酸生產之總量 39

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